Arterial replacement during surgical procedures has employed the use of prosthetic grafts as an alternative to actual vein replacements. These prosthetic grafts are made from such materials as expanded-polytetrafluoroethylene (ePTFE), polyacrylonitrile polymers, polystyrene, polyester and polyethylene terephthalate. Although the use of synthetic grafts have been successful for the replacement of larger vessels such as the aorta, synthetic graft replacement has often been unsuccessful in the replacement of smaller vessels. The absence of a functional, non-thrombogenic endothelial cell monolayer on the luminal surfaces of small diameter prostheses appears to be a major factor limiting long term patency. Therefore, it is desirable prior to implantation of prosthetic grafts that a seeding of endothelial cells onto the luminal surface takes place.
Because the number of human endothelial cells that can be harvested from the limited lengths of donor vessel available is low, it is desirable to develop methods for enhancing cell attachment and subsequent spreading of these cells. The aim of this type of research is to optimize the formation of a functional, non-thrombogenic endothelial cell monolayer by improving cell attachment onto the inner surface of graft material using various surface coatings. See Budd, J.S., et al., Br. J. Surg. 76:1259-1261 (1989). Another related aim of this type of research is to develop methods for introducing various substances and products into the blood system by the seeding of genetically-altered endothelial cells.
Shortcomings associated with replacement vascular grafts have included disappointing graft endothelialization, thus significant luminal thrombus formation resulting in poor long term patency. In addition, the formation of a confluent endothelial cell monolayer on the graft following implantation has been poor, probably due to significant cell losses from the graft surface resulting from the stresses of pulsatile blood flow. Prior studies have indicated that the formation of a firmly attached endothelial monolayer on the inner graft surface before implantation provides a much more stable base for the duplication and spreading of cells following implantation which leads to non-thrombogenicity and permanence of the graft.
Endothelial cell seeding on the inner surface of synthetic grafts has been the focus of research efforts toward producing synthetic grafts which are essentially non-thrombogenic. It has been noted that the use of certain growth media significantly improves the initial attachment of endothelial cells onto synthetic grafts in a static system. See Budd, J.S., et al., Br. J. Surg. 78:878-882 (1991).
Known methods for cell seeding on synthetic grafts have included coating the luminal graft surface with an enhancing substance, such as fibronectin, before introducing a cell suspension into the graft. The cell suspension commonly includes endothelial cells that are harvested from human veins by flushing into a first suspension and centrifuging to produce a cell pellet. This cell pellet is resuspended in a growth medium to produce a cell suspension suitable for introduction into the synthetic graft. The synthetic graft containing the cell suspension on its luminal surface is incubated within an incubation chamber for a period of time to allow attachment of endothelial cells to the inner surface of the graft. In this incubation procedure the ends of the graft are sealed to keep the cells and medium inside, and provide a favorable cell growth environment, preventing the evaporation of the cell growth medium from within the synthetic graft. The synthetic graft is also slowly rotated during the incubation period to provide an even distribution of endothelial cells throughout the inner surface of the graft material by evenly distributing the cell suspension.
Known methods for sealing the ends of a synthetic graft have included the use of rubber or plastic plugs or clips, clamps, sutures or glass plugs with a syringe fitting at one or both ends. The use of these external sealing means, however, can be cumbersome and inconvenient, especially when sealing synthetic grafts of small diameters. In addition, these external means for sealing the ends of synthetic grafts can fail to provide graft ends that are completely closed or can become separated from the graft by changes in temperature, pressure or orientation of the graft, as well as by movement of the graft associated with its handling.
Known methods for culturing cells within an incubation chamber have included the insertion of an EPTFE piece inside a glass bottle with TEFLON.RTM. tubing attached at each end. The EPTFE piece in this arrangement was taped to the inside surface of the bottle. James et al., Artif. Organs 14:355-360 (1990). In another method, a glass tube, itself a substitution for EPTFE, was used as a cell growth chamber with cells growing on the inside surface of the glass tube. This tube was inserted into a rotation device. Int. J. Artif. Organs 12(4): 270-5 (1989). Many of the known methods are cumbersome, however, in that they utilize either a large number of tubes within a single roller bottle, or employ a single mechanical rotating device for rotating only a single tube.
The disadvantages associated with these methods have included the failure of the graft to be separated from the interior surface of the surrounding tube or bottle for purposes of maintaining uniform temperature and humidity. Also, when the external diameter of the synthetic graft is substantially smaller than the internal diameter of the glass or plastic tube or bottle surrounding the synthetic graft in the rotation device, the repetitive loss of frictional contact between the external graft surface and the internal test tube surface with rotation of the tube or bottle can result in a repetitive sliding of the synthetic graft within the tube or bottle during rotation by a rotation device. Such movement of the graft within the tube or bottle is undesirable because it can be associated with sudden jarring movements at the point of friction loss and can therefore cause a detachment of endothelial cells from the inner surface of the graft.
The need therefore exists for a method for sealing the ends of a synthetic graft conveniently and substantially, without the need for external sealing means. The need also exists for separating the graft with respect to a rotation container during incubation, such that the synthetic graft rotates smoothly in response to rotation of the rotation device without any slipping or jarring caused by loss of frictional contact between the external graft surface and the internal test tube surface.